Download Superior Pointing Stability Simplifies MPE Imaging

White Paper
Superior Pointing Stability Simplifies MPE Imaging
A clever optical design, superior component
tolerance, rugged engineering, and rigorous factory
testing ensure the pointing stability of the
Chameleon™ Vision is virtually twice as good as
any other dispersion compensated MPE laser
source, avoiding any need for tweaking the
alignment between the laser and MPE microscope.
Introduction and Overview
Multiphoton excitation (MPE) applications have become
demonstrably easier since one-box ultrafast lasers
made available an ideal combination of optimized
performance and operational simplicity. And because
MPE microscopes are often shared resources, users
also derive considerable benefit from the wide tuning
range of these hands-free lasers – 680 nm to 1080 nm
in the case of the Coherent Chameleon series. For
instance, the long wavelength is particularly useful for
optimum excitation of red fluorophores such as mcherry. And more recently, the integration of smoothly
and widely variable group velocity dispersion (GVD)
compensation of up to 47,000 fs2 – enables the final,
at-sample, pulsewidth to be varied and/or minimized at
will, even for next generation microscopes with multiple
AO modulators. Although all this useful functionality is
now provided with push-button simplicity, it’s very
important that its implementation does not compromise
beam pointing stability, either during normal use or
when a laser is moved. That’s because beam pointing
stability is critical to maintaining downstream alignment
between the laser and the microscope. Without
adequate pointing stability, changes in the laser output
parameters could necessitate re-alignment of the beam
into the microscope, negating the very simplicity and
experimental efficiency these lasers were originally
devised to provide. In this whitepaper we detail the
design and construction factors which lead to superior
alignment for the Chameleon Vision.
Microscope Alignment
In a MPE microscope, the input alignment is very
important. The most critical alignment is with the back
aperture of the objective. Ideally, the beam is centered
on this aperture and overfills it so that the laser beam is
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close to a plane wave. Any misalignment manifests
primarily as lost intensity at the sample. However,
since the beam intentionally overfills this aperture,
some beam walk-off can be tolerated. How much?
The exact specifics depend on the particular
microscope model, but as a rule of thumb, a laser
pointing deviation of 400 microrad is a typical maximum
value that a MPE microscope can tolerate before the
at-sample intensity drops noticeably. If the deviation is
higher than this then the operator will have to tweak the
steering mirrors between the laser and microscope,
which is a very undesirable scenario for a highthroughput resource often shared by several scientists
who may not all be experts in optical alignment.
Defining Performance
In a well-designed integrated oscillator, the primary
source of potential pointing deviation is wavelength
tuning, since the refractive indices of the cavity optics
vary with wavelength. So the critical specification is
beam pointing as a function of output wavelength. The
Chameleon Vision has a specified pointing stability of
80 microrad per 100 nanometers of tuning.
Figure 1: The Chameleon Vision has a specified pointing stability
of 80 microrad per 100 nanometers of tuning. As shown by this
test data, the typical performance is normally far better than this
guaranteed worst-case specification.
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That means that even for 400 nm tuning, the maximum
pointing movement is only 320 microrad, which is well
below the 400 microrad threshold where intensity
effects can be noticed in typical microscopes.
Moreover, the typical performance is normally far better
than this guaranteed worst-case specification, as
shown in Figure 1.
Indeed, the Chameleon Vision currently has the best
pointing stability specification for any dispersion
compensated MPE laser. But when comparing MPE
lasers, it is very important to understand how the
pointing is being specified. The microscope input
aperture has a circular cross section, so at Coherent,
we use a radial specification, i.e. 80 μrad/100 nm in
any axis. That’s because with a circular aperture, it
doesn’t matter which direction the beam walks – if it’s
more than 400 microrad, it can be a problem.
However, some MPE lasers are specified according to
performance in orthogonal axes, which is somewhat
different. For example, a laser specification of 100
μrad/100 nm in each (xy) axis is not the same as a
radial specification of 100 μrad/100 nm. To understand
this, consider a worst-case scenario for both
specifications. 80 μrad/100 nm in any axis means just
that – this is the maximum beam deviation any axis, i.e.
320 μrad for 400 nm tuning. But for a specification of
100 μrad/100 nm in each (xy) axis, the maximum
deviation is 100 μrad in the x axis and 100 μrad in the y
axis, giving a maximum radial deviation of √20,000, i.e.
141 μrad (see Figure 2). So what appears to be only a
20% difference in specification is actually almost a
factor of 2, with a worst case scenario of 565 μrad for
400 nm of tuning.
Stability by Design
How does the Chameleon Vision deliver this superior
performance? The key is in the design and assembly
as well as rigorous testing. This laser is inherently very
stable in terms of beam pointing, which is preferable to
the alternative of inferior performance and active
compensation. The latter would add unnecessary cost
and complexity.
One way to achieve passive stability in an optical
system with an adjustment involving angular rotation is
to use a retroreflector. These are prisms or mounted
mirror assemblies that return an incident light beam
back along an axis that is perfectly parallel to the
incoming light path. Where the beam is large enough
to fill the input aperture of a retroreflector, the return
beam retraces the path of the incident beam.
Obviously, system performance depends on
manufacturing the retroreflector(s) to extremely high
angular tolerances. Extensive testing and evaluation in
Coherent’s R&D laboratories has shown that the best
solution for an MPE laser is to use a different type of
retroreflector, the rooftop or Amici prism (see Figure 3).
These are right angle (90°) prisms that provide a
retroreflection parallel in one plane only. Using two of
these prisms allows any slight walk-off in both x and y
axes to be avoided, yet without specifying trihedral
prisms or hollow retroreflectors optics with tolerances
that would be very difficult (and prohibitively expensive)
to consistently produce. Nonetheless even using
rooftop prisms requires custom optics, designed and
fabricated at Coherent – no commercial catalog prisms
offer the necessary tolerance.
A roof prism reflects
a beam back along its
incident angle, no matter
what the input angle
Figure 2: The Chameleon Vision delivers a specified pointing
stability better than 80 μrad/100 nm in any axis. For comparison,
we show a specified pointing stability of 100 μrad/100 nm in x and
y axes, where the worst case beam deviation is actually 141
μrad/100 nm.
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Figure 3: When used as a retroreflector, a rooftop prism returns
an incident beam back along its original direction. This
automatically corrects for any angular walk-off due to rotation of
optical elements in the beam path.
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Of course, opto-mechanical stability is also important,
particularly in a compact one-box laser intended to be
easily moved from bench to bench or even lab to lab.
Again, Coherent has opted for inherent passive stability
over complex and costly active correction. This
includes extensive use of our patented PermAlign™
mounting method for critical optics, all the way through
the system from the green Verdi™ pump laser to the
final GVD pre-compensation module as well as
between the Ti:Sapphire oscillator output and this
module. With PermAlign, each optic is precisely
aligned and then soldered into a one-piece mount
which is rigidly bolted to the laser baseplate. With no
adjustment screws, there is no possibility of such a
mount being shifted out of alignment by thermal
variations or by mechanical vibration or shock. This is
the reason that Chameleon Vision can be moved
around an optical table or transported from one lab to
another without any degradation in the alignment
between the Ti:Sapphire oscillator and the GVD
module. With competitive designs using conventional
multi-part mounts, this is frequently not the case.
With every single Chameleon Vision we build, we want
to find any stability problems so that you don’t. This
includes environmental testing and cycling of each
laser before shipping, under conditions that truly
represent conditions beyond the worst case the laser
can be expected to have to endure during its lifetime.
Where necessary, Coherent engineers analyze and
identify the root cause and correct it. Key aspects
include temperature, humidity and vibration cycling –
often referred to by quality engineers as “shake and
bake.” The laser output is continuously monitored
during these tests to check that its performance never
deviates beyond guaranteed specifications.
Conclusion
The Chameleon Vision is a high-performance laser
source for many types of MPE microscopy, providing a
superior wavelength tuning range as well as the highest
amount of adjustable GVD compensation. And due to
its unrivalled beam stability characteristics, it’s also a
high-throughput source designed to ensure you’re time
is spent taking images and other data, and not spent
tweaking the alignment as imaging conditions change.
The final element enabling Chameleon’s superior
specifications is comprehensive testing. As famously
demonstrated by the initial problems with the Hubble
telescope, this requires full testing of the entire system
operating in its intended mode.
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